Creating Nanoparticles with
Microfluidizer® High-Shear Fluid
Technology
Yang Su
New Technology Manager
Microfluidics International Corporation
1
Microfluidics at a Glance
• Microfluidics has been located just outside of Boston,Status
MA forUpdate
32 years serving
over 2000 customers worldwide. We have sold ~4,000 processors with
localized sales and support in 47 countries.
• Microfluidizer® high shear fluid processors can produce nanomaterials with a
wide variety of multiphase applications. We have vast experience with process
development of many different types of applications/formulations. We pride
ourselves in our ability to help our customers get the most out of their
materials.
• Microfluidizer Processors are used for R+D and manufacturing of active
pharmaceutical ingredients, vaccines, inkjet inks, coatings, nutraceuticals and
cosmetics.
17 of the top 20 pharma companies
8 of the top 10 biotech companies
4 of the top 5 chemical companies
...innovate with Microfluidics technology
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What Microfluidics Does Best
•
•
•
•
•
•
•
Nanoemulsions
Cell disruption
Protein recovery
Uniform particle size reduction
Nano/microencapsulation
Nanodispersions
Deagglomeration
M-110P “plug n’ play”
benchtop lab model
M-110EH-30
pilot scale machine
“The overall satisfaction which
we experienced with our
laboratory model Microfluidizer
processor eliminated the need
to consider other equipment
when it was time to scale up to
production capabilities.”
- Amylin Pharmaceuticals
M-700 series
production machine
Fixed-geometry
interaction chambers
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Microfluidizer® Processors & Interaction Chamber (IXC)
•
•
•
•
Microchannels
range from 50-500 microns
Continuous processing
•
High pressure used to• deliver
product
the
interaction
•
Unsurpassed shear and impact forces
Accommodates materialsinto
with
high
solid chamber
content
•
Constant pressure pumping
system
•
Temperature
regulated by a heat exchanger
Can accommodate high viscosities
•
Lack of moving parts maximizes uptime
Can work in a wide range of temperatures
•
Repeatable and scalable results
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Microfluidizer® Processors & Interaction Chamber (IXC)
• Exceptional performance – Channel velocities over 400 m/s and generate shear
rates up to 108 s-1
• Consistent processing – Fixed geometry with no moving parts and guaranteed
scalability
• Long-wearing – Made from diamond or ceramic materials
• Ease of maintenance – Clean-in-place and steam-in-place
• Many options available – Variable shape and size
Scale Up
Scale Up
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Constant Pressure and Constant Volume
• Virtually all of the product is passed through the Microfluidizer processor at the
target pressure.
• Only a small portion of the product is passed through the high pressure
homogenizer at the target pressure.
Microfluidizer processor
Homogenizer Valve
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Shear Rates for Various Technologies
Microfluidizer
Processor
Homogenizer
RotorStator
Sawtooth
Blade
Agitator
Colloid
Mill
Closed
Rotor
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Nominal Shear Rates as a Function of Pressure & Chamber
Data shown for internal diameters vary from 75 m – 300 m
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Benefits of Microfluidizer® Processors
• How Microfluidics Technology
is Unique
LV1
1 mL hold up volume
• Resulting Benefits
Scale Up
> Constant Pressure Processing
> High Potential Processing
Pressures
> Fixed Geometry Interaction
Chambers
> Multi-Slotted Interaction
Chambers
> Very small particle size potential
> Very consistent processing
resulting in very narrow particle
size distributions
> Guaranteed scale-up from lab
scale to production scale
• cGMP Compliance and CIP/SIP
Capabilities
M7250-20 Pharmaceutical/
Constant Pressure/SIP
Can process 8 LPM at 1300 bar
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Nanoemulsion
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Nanoemulsion
• Nano-emulsions are formed when two or more
immiscible liquids are mixed and one phase is
finely dispersed in the other (s)
– Oil-in-water
– Water-in-oil
– Double emulsions (O/W/O, W/O/W)
• Surfactants stabilize the emulsions by
decreasing the surface energy between the
immiscible liquids; they can be ionic, polymeric,
proteins or solid particles.
• Oil-in-water nanoemulsions are used for
delivering water insoluble active
pharmaceutical ingredients (APIs).
Discrete phase
surrounded by surfactant
Continuous phase
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Nanoemulsions as Vaccine Adjuvants
• Vaccines are biological preparations used to improve immunity to a
particular disease
• Vaccine adjuvants are materials that enhance the efficacy of vaccines
• Most new vaccine adjuvants are emulsions or liposomes
– Effective: Enhance both cellular (Th1), humoral (Th2) and major
histocompatibility complexes (MHC) responses
– Well tolerable
– Biodegradable
Inflammasome
activation (Alum)
Immune cell/
MHC presentation
(Emulsions)
Depot effect
Adjuvant
PRR activation
(TLR/NOD agonists)
Th1 cell
Th2 cell
Opsonizing
antibodies
Neutralizing
antibodies
Antigen
www.invivogen.com/review-vaccine-adjuvants
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Nanoemulsion
• Nanoemulsion production challenges:
– Stability
• The presence of particles over 1 micron may destabilize the
emulsion through Ostwald-ripening
– Sterilization
• Removal of bacteria by filtering though a 0.22 micron rated filter
• Preferred sterilization method in vaccine adjuvants, cancer drugs
(injectables)
• Most particles should be below 0.22 microns so they do not plug
the filter
Asymmetric
Symmetric
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Nanoemulsion – Vaccine Adjuvant Nanoemulsion
• Nanoemulsions are promising new vaccine adjuvants
• Squalane Emulsion
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Nanoemulsion – Vaccine Adjuvant Nanoemulsion
Collaboration with Pall Life Sciences
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Nanoemulsion – Ocular Nanoemulsion
• An ophthalmic nanoemulsion used to treat dry eyes
• Castor oil in water nanoemulsion
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Nanoemulsion – Anesthesia Nanoemulsion
• Used for induction and maintenance of anesthesia
• Soybean oil in water nanoemulsion
Unprocessed
Particle Size (μm)
Pressure
(psi)
#
Passes
D10
d50
d90
Unprocessed
0
0.954
13.678
24.201
1
0.200
0.290
0.401
2
0.187
0.247
0.323
3
0.134
0.181
0.244
20,000
3 passes
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Cell Disruption
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Cell Disruption
• The generation of important enzymes, proteins and other
products form microbes has been developed and used for
the last 40 years
• Cell rupture is required any time that products from cell
sources must be removed from inside the cell
• Recover the most product
– Rupture the highest percentage of cells
– Minimize the potential for denaturing the protein (Shear,
Temperature, etc.)
• Microfluidizer is a well known “Technology” within the
biotech industry
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Cell Disruption
• Bacterial cells are the most commonly used
cells for production of simple proteins
– Usually only require 1P on a Microfluidizer® to
achieve >90% rupture efficiency
Source: NIAID, NIH
• Yeast cells have the benefit of creating complex
proteins
– Among the hardest to rupture: high shear and
multiple passes
Source: Johns Hopkins Univ.
Mammalian
Bacteria
Yeast
Algae
0
1
2
3
5
4
Shear rate (s-1 X 106)
6
7
8
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Cell Disruption – E.coli
• Optimization of Eschericia coli W3110 pTrcHisB:opd cell
disruption and Organophosphate Hydrolase recovery
• Grow cells in a 2L bioreactor
• Rupture cells at various conditions:
– 1 and 2 passes
– Three different pressures
• Enzyme activity assay after IMCA purification
– Determines activity of target enzyme vs. Bradford assay which
determines total protein recovery
• TEM analysis of cells before and after rupturing
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Cell Disruption – E.coli
Unprocessed
1 Pass
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Cell Disruption – Yeast (Pichia Pastoris)
Process conditions: 1 pass 30,000 psi (2070 bar)
Chamber: H10Z (100 microns)
Shear rate: 6.94 X 106 s-1
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Cell Disruption – Yeast (S. pombe)
Process conditions:
30,000 psi (2070 bar)
Chamber:
G10Z (87 microns)
Shear rate per pass:
7.37 X 106 s-1
Unprocessed
1 pass ~60% lysis
5 passes ~95% lysis
10 passes ~99% lysis
Maximum soluble
protein recovery
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Size Reduction of Solid API Suspension
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Size Reduction of Solid API Suspension
• Many new APIs are “poorly water soluble”
• Typical time that actives will stay in the body are 12-18
hours.
• In order to achieve therapeutic doses, large quantities of
active must be administered.
• Active ingredients that are not dissolved are typically
removed by the liver
• Particle size and size distribution reduction
– Does not change the solubility
– Changes (increases) surface area, which greatly affect the
dissolution rate and in most cases the bioavailability
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Size Reduction of Solid API Suspension – Atovaquone
• Used to treat many conditions
including
>
>
>
>
Malaria
Toxoplasmosis
Babesia
Pneumocystis pneumonia (PCP)
Source: lymewithpurpose.wordpress.com
• Generally used in combination
with other drugs
> Proguanil
> Azithromycin
• It prevents the electron
transport of enzymes
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Size Reduction of Solid API Suspension – High Blood Pressure Drug
1 pass
Median particle size (D50) after 1 pass: 955 nm
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Frequency (volume%)
Size Reduction of Solid API Suspension – Epilepsy Drug
12
Unprocessed
10
8
6
4
2
0
0.01
0.1
1
10
100
1000
100
1000
Particle Size (microns)
Frequency (volume %)
8
7
6
Industrial
Lab
5
4
3
2
1
0
0.01
0.1
1
10
Particle Size (microns)
• Median particle size (D50) with lab machine: 773 nm
• Median particle size (D50) with production machine: 614 nm
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Liposome
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Liposome
• Liposomes are spherical lipid vesicles with
a bi-layered membrane structure
• Can encapsulate either hydrophobic or
hydrophilic active, or both
• One of the most successful delivery
systems currently in clinical use
Hydrophobic active
Hydrophilic active
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Liposome – Liposomal Active for the Treatment of Cancer
Company: A large US based pharmaceutical company
Application: Treatment of carcinomas
Material: A poorly water soluble active pharmaceutical ingredient with a melting
point of < 60ºC
Goal: To analyze various processing conditions and formulations to produce
liposomal formulations of the active in the range of 50 nm to 150 nm.
Frequency (vol. %)
Results:
16
14
12
10
8
6
4
2
0
10
100
1000
Particle Size (nm)
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Polymer Nano-Suspensions
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Polymeric Nanoparticles
• Combination drug products
• Drug and resistance modulator
• Drug and energy delivery (heat, light,
and sound)
• Drug and imaging agent
Therapeutic/imaging
payload
Polymer
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Polymer Nanoparticles – Emulsion Evaporation Method
Polymer
Solvent
Water
Surfactant
Drug
STEP 1
Oil
Phase
+
Water
Phase
Microfluidizer
Processor
Nanoemulsion
Solvent
Removal
STEP 2
Nanoemulsion
Purification
Nanosuspension
• Solvent is not miscible with water
• Post processing is critical and includes removal of solvent and the formation of nanosuspensions
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Polymer Nanoparticles – Emulsion Evaporation Method
• Polymer/solvent/non-solvent system: poly(lactic-co-glycolic acid (PLGA) /
dichloromethane (DCM) / D.I. water
Z-avg. particle size (nm)
• Processed with M-110EH for 1 pass at 10,000psi (70 Mpa)
250
Polymer
particle
225
200
Contrasting
agent (PTA)
175
500 nm
150
0
50
100
Conc. of PLGA in DCM (mg/ml)
TEM images were taken to
verify particle size
T. Panagiotou, S.M. Mesite, J.M. Bernard, K.J. Chomistek and R.J. Fisher, NSTI-Nanotech 2008, ISBN 978-1-4200-8503-7 Vol.1
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Polysaccharide Molecular Weight Reduction
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Polysaccharide Uses in Pharmaceuticals
• Polysaccharides are utilized in many pharmaceutical applications
– Biocompatible and biodegradable in the body
– Naturally broken down to building blocks, allowing for drug release without
inflammatory immune response
• Polysaccharides have diverse molecular weights (MW) and
structures
– Hydrophilic groups enable bond formation with tissues and mucosal
membranes, and extended circulation in blood
→ increased probability of targeting of tumors
– Hydrophobic groups also prevalent on polymer backbone
→ good carriers for water-insoluble drugs
– Low MW polysaccharides show positive Zeta
potential and higher solubility at a neutral pH
Exercise physiology: energy, nutrition, and human
performance. Lippincott Williams & Wilkins. 2006
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Polysaccharide MW Reduction
• Material: Polysaccharide vaccine recently developed to enhance
production of type-specific antibodies against pneumococcus in
high-risk patients
• Goal: Reduce molecular weight of the polysaccharide to below
250 kDa
• The LV1 was utilized to process polysaccharide formulations
1000
(k D a )
800
MW
 Results indicate that the MW of
7 out of 8 of the polysaccharides
were reduced below the targeted
MW using the Microfluidizer®
S E C - H P L C S iz in g r e s u lt s
600
1
7F
6B
9V
14
5
19A
23F
19F
400
200
0
0
5
10
15
20
M F P assage N o.
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Summary
• Microfluidizer high shear fluid processors can produce
nanomaterials with a wide variety of multiphase applications
• Through the demonstrated case studies, MicrofluidizerTM
technology has been proven to:
– Be superior to conventional technology.
– Be very efficient and reliable.
– Offer precise, repeatable and scalable results.
• For biopharma industry, production Microfluidizers® are
designed for compliance with cGMP and are capable of CIP/SIP
operations.
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Any Questions?
I will be happy to address any questions
Yang Su
Microfluidics International Corporation
617.969.5452 x 320
[email protected]
Learn More
Visit www.microfluidicscorp.com to view our complete technology
capabilities, or submit a sample for Proof of Concept testing
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